The use of proteins as binding partners for the detection of analytes in immuno-diagnostic test procedures has been known for a long time. In all conventional immunoassays, the sample is incubated with one or more binding partners that are specific for the analyte. The binding partner or binding partners bind(s) specifically to the analyte to be detected. In the case of an antibody test, for example, in the case of an HCV infection, the sample to be examined is, for example, contacted with an HCV antigen which specifically binds the anti-HCV antibody to be detected. In an antigen test, for example, for detecting the tumour marker prostate-specific antigen (PSA), the sample is contacted with antibodies which specifically bind the PSA in the sample.
Subsequently the analyte is detected in all immunoassays. This can, for example, be carried out by binding and subsequently detecting another binding partner provided with a detectable label which binds to the complex consisting of analyte and immunological binding partner.
In general the immunoassays are carried out in a heterogeneous or homogeneous test format. The heterogeneous test formats are frequently carried out as sandwich or bridge tests. Competitive methods are also well known in which either the analyte or the specific binding partner is displaced from the complex of analyte and specific binding partner by, for example, adding a labelled analyte analogue.
In all immunological test methods, it is important that the reactants used as the specific immunological binding partners are present in a stable form and that they are not destroyed, for example, by unfavourable storage conditions. This risk can occur in particular when the proteins used as specific binding partners are composed of several subunits. The subunits can be held together covalently, for example, by means of disulfide bridges, or non-covalently, for example, by means of hydrogen bridges, opposite charges, and/or hydrophobic interactions.
In some cases, the materials required for the immunological test may become unstable and denature under the storage conditions (for example, as a liquid reagent) in the working solutions prepared for the test or during the immunological reaction itself. As a result, the tertiary and the quaternary structure of the protein may be changed in such a manner that the substance can no longer be used in the immunoassay.
The subunit components of the proteins used as a specific binding pair may separate under unfavourable conditions. This dissociation of subunits may, for example, be caused by the reduction of disulfide bridges by common buffer additives such as DTT in the case of natural covalent bridges.
However, the risk of dissociation is even higher in the case of non-covalently linked subunits of a protein which are held together by charges or hydrophobic interactions. The subunits of such proteins can be very easily dissociated even by common buffer additives such as salts, detergents, or unfavourable variations in pH and temperature. An individual and hence unprotected subunit is thus also susceptible to denaturation. This may lead to major changes in the tertiary structure of the protein or of the individual subunit. This also means that the immunological properties such as the accessibility of important epitopes is changed to such an extent that the protein used as a binding partner in the immunoassay is no longer recognized immunologically and is hence no longer specifically bound.
Another risk of subunit dissociation is that subunits provided with different labelling groups may re-associate due to the adjustment of the chemical equilibrium. If in a specific case, a protein composed of two subunits for use in an antibody test in a bridge test format is derivatized in order to be used as a universal solid phase and, on the other hand, the same protein is also used as a signal-generating component and for this purpose is coupled to a label (e.g., an enzyme, fluorescent label, or chemiluminescent label), the following may happen: a calibration curve which is initially generated with positive samples (samples which contain the analyte) becomes flatter as time progresses. The signals for negative samples (blank values) increase and increasingly approximate the values for the upper positive samples so that it is no longer possible to differentiate between analyte-free and analyte-containing samples.
A method for chemically modifying enzymes by reaction with quinones is described in German Patent Application DE 26 15 349. These modifications increase the stability which results in an improved enzyme activity. It is mentioned that the enzyme molecules can be cross-linked to one another, i.e., intermolecularly and also intramolecularly. In this case, the preservation of immunoreactive epitopes is irrelevant. The use of enzymes modified with quinones in immunodiagnostic methods is not described.
Debyser and De Clercq (Protein Science 5, p. 278-286, 1996) describe the cross-linking of the two subunits of HIV-1 reverse transcriptase by means of dimethyl suberimidate which cross-links lysine side chains. The purpose of the cross-linking is to examine the dimerization of the two RT subunits. Only the dimeric RT is enzymatically active. The two subunits are covalently cross-linked in the presence of various inhibitors. RT molecules and multimers that are more or less strong cross-linked depending on the effectiveness of the inhibitor are formed after the chemical cross-linking reaction. The effect of the cross-linking on immunologically relevant epitopes or the use of cross-linked molecules in immunoassays is unimportant.
The use of intermolecularly cross-linked immunoglobulins in immunoassays is disclosed in EP-A-0 331 068. This means that several immunoglobulin molecules or fragments thereof are covalently linked together. The multimers of antibodies and fragments thereof are used as an interference-reducing reagent. The cross-linked immunoglobulins and fragments thereof are intended to eliminate interfering factors of human serum that are directed towards immunoglobulins.
The cross-linked proteins described in the prior art, which are composed of several subunits under natural conditions, are unsuitable or of only limited suitability for use as antigens or immunological binding partners since, in general, intermolecular multimers consisting of several protein molecules are formed. These multimers are of only limited use for immunoassays since they usually do not have a defined size. Hence the multimers have a random distribution of sizes, i.e., mono-, di-, tri-, tetramers, etc. are present together in one mixture. The undefined cross-linking may mask the epitopes. Consequently a sample antibody to be detected may not be able to bind to the masked epitope of the antigen, and hence a false negative result is obtained.
Another problem with using multimers as immunological binding partners is the fact that there is an increased risk that interfering factors present in the sample may bind unspecifically to the multimeric proteins. Interfering factors such as rheumatoid factors often have several binding sites of low affinity. If multimeric proteins are then used as immunological binding partners, this may have the effect that especially the interfering factors find many targets, i.e., binding sites on the multimeric proteins. This may lead to false positive test results, and the overall specificity of the immunoassay is greatly reduced.